4-Oxo-1,4-dihydropyridines as selective CB2 cannabinoid receptor ligands part 2: Discovery of new agonists endowed with protective effect against experimental colitis

Article abstract of DOI:10.1021/jm3008568

Further on to our earlier work on the 4-oxo-1,4-dihydropyridine, we describe herein our strategy to get access to potent selective CB₂ receptor agonists. Thus, we designed and synthesized 29 compounds, evaluated on both hCB₁ and hCB<

Full text of DOI:10.1021/jm3008568

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Brief Article
4-Oxo-1,4-dihydropyridines as Selective CB2 Cannabinoid
Receptor Ligands Part 2: Discovery of New Agonists
Endowed with Protective Effect Against Experimental Colitis
Jamal El Bakali, Pauline Gilleron, Mathilde Body-Malapel, Roxane Mansouri, Giulio G Muccioli,
Madjid Djouina, Amélie Barczyk, Fréderique Klupsch, Virginie Andrzejak, Emanuelle Lipka,
christophe furman, Didier M Lambert, Philippe Chavatte, Pierre Desreumaux, and Regis Millet
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Sep 2012
Downloaded from http://pubs.acs.org on October 8, 2012
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Journal of Medicinal Chemistry
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4-Oxo-1,4-dihydropyridines as Selective CB2 Cannabinoid Re-
ceptor Ligands Part 2: Discovery of New Agonists Endowed
with Protective Effect Against Experimental Colitis.
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Jamal El Bakali,1 Pauline Gilleron,1 Mathilde Body-Malapel,2 Roxane Mansouri,1 Giulio G. Muc-
cioli,3 Madjid Djouina,2 Amélie Barczyk,1 Frédérique Klupsch,1 Virginie Andrzejak,1 Emmanuelle
†
Lipka, 4 Christophe Furman,5 Didier M. Lambert,3 Philippe Chavatte,1 Pierre Desreumaux,2 and
Régis Millet.1*
1 Institut de Chimie Pharmaceutique Albert Lespagnol, Université de Lille Nord de France, E.A 4481, IFR 114, 3 rue
du Pr. Laguesse, B.P. 83, F-59006 Lille Cedex, France
2 Digestive inflammatory diseases: pathophysiology and development of therapeutic targets. U995 INSERM, Uni-
versité Lille Nord de France, IFR 114, Amphis J & K, Boulevard du Professeur J. Leclercq, 59045 Lille Cedex, France
3 Unité de Chimie Pharmaceutique et de Radiopharmacie, Louvain Drug Research Institute, Université catholique
de Louvain, 73 avenue E. Mounier UCL-CMFA (7340), B-1200 Bruxelles, Belgium
4 Laboratoire de Chimie Analytique, EA4481, IFR 114, 3 rue du Pr. Laguesse, B.P. 83, F-59006 Lille Cedex, France
5 Laboratoire de Toxicologie - EA 4483, Université de Lille Nord de France, F-59000 Lille, France.
ABSTRACT: Further to our earlier work on a the 4-oxo-1,4-dihydropyridine, we describe herein our strategy to get access
to potent selective CB2 receptor agonists. Thus, we designed and synthesized twenty nine compounds, evaluated on both
hCB1 and hCB2 cannabinoid receptors and assessed eleven of them in the TNBS-induced colitis model in mice. Com-
pound 48 was found to be the most efficient of our series, exhibiting an exquisite protection against experimental colitis,
superior to the one observed after treatment with Pentasa.
CB1/CB2 agonists (Δ9-THC, 1 and HU-210, 2)8 as well as CB1
INTRODUCTION
(Arachidonyl-2'-chloroethylamide, 3)9 and CB2 (JWH133, 4
and AM1241, 5)5,9,10 selective agonists were shown to sig-
CB2 cannabinoid receptor is a G-protein coupled recep-
tor that constitutes, with CB1 cannabinoid receptor, the
nificantly reduce colon alterations in several experimental
restricted family of cannabinoid receptors (CNRs), the
models of IBD in rodents. Similar results were obtained
two main targets of endocannabinoids (ECs).1 The latter,
when using ECs membrane transport inhibitor 6 (VDM11)
including N-arachidonoylethanolamine (anandamide,
as
well
as
FAAH
(5-([1,1'-biphenyl]-4-yl)-N-(2-
AEA) and 2-arachidonoylglycerol, are lipid mediators that
are biosynthesized on demand, and which, by their action
on CNRs, exert a variety of physiological responses, in-
cluding in the gut.2 All the components of the endocan-
nabinoid system have been identified in both normal and
inflamed gastrointestinal (GI) tract. The CB2 receptor is
widely expressed in immune cells, such as macrophages,
B and T cells,3 which are involved in the inflammatory
response during colitis.4 In addition, it has been shown to
be up-regulated in the inflamed colon,5 while a reduction
in FAAH expression accompanied by an elevation of the
AEA levels has been reported.6 These events have been
hypothesized to counteract abnormal gut inflammation,
and hence to protect against colitis. Therefore, the endo-
cannabinoid system, including CB2 receptors, emerged as
promising therapeutic target for the treatment of GI tract
inflammatory conditions. Over the past few years, phar-
macological modulation of both CNRs and ECs hydrolyz-
ing enzymes were shown to protect against colitis.7 Dual
(benzo[d][1,3]dioxol-5-yl)ethyl)isoxazole-3-carboxamide, 7
and URB597, 8)11 and monoacylglycerol lipase (JZL184, 9)
inhibitors.12 Unfortunately, some of these ligands are well-
known to exert undesirable psychotropic effects that are
assumed to be central CB1 receptor-mediated. According-
ly, CB2 receptor selective agonists, with their non-
psychoactive profile, are likely to have therapeutic value
in the treatment of IBD.
Therefore we aimed to develop new CB2 selective ago-
nists endowed with anti-inflammatory properties in the
GI tract. Along this line, we recently described a series of
4-oxo-1,4-dihydropyridines as CB2 receptor selective lig-
ands.13 We showed that the C-6 substituent was able to
govern the functional activity of these compounds, which
act as agonists or inverse agonists depending on the pres-
ence of alkyl or aryl groups, respectively, at C-6 position.
This work suggested that it was conceivable to design CB2
ligands, based on a 4-oxo-1,4-dihydropyridine scaffold,
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with a predictable functional activity. These crucial find- derivatives. Contrasting with this, compounds character-
1
2
3
4
5
6
7
8
ings gave us the opportunity to obtain CB2 selective ago-
nists of therapeutic interest in the treatment of colitis.
ized by a methyl at C-6 and presenting a bulky aliphatic
group at the amide function (30-33) displayed very high
affinity for the CB2 receptor while being devoid of CB1
receptor affinity. Compound 32, characterized by a 2-
adamantyl moiety, presented the best profile in our series
with regard to affinity at CB2 receptor and selectivity over
CB1 (hCB2: Ki = 0.26 nM and S.I > 3846).
RESULTS AND DISCUSSION
Preliminary in vivo Studies. We first evaluated our
lead CB2 selective agonist 10 (N3-(1-Adamantyl)-6-tert-
butyl-1-pentyl-4-oxo-1,4-dihydropyridine-3-carboxamide,
hCB2: Ki = 29 nM and hCB1: Ki > 3000 nM)13 in the TNBS-
induced colitis model in mice (Chart 1).14 Pentasa formu-
lation of 5-aminosalicylic acid was used as positive control
(150 mg/kg). After being administered once daily (10
mg/kg, i.p.), 10 was able to decrease colitis macroscopic
scores by 19.3% (80.7% vs 100,0%, p=0.04), thus having
the same effect than our positive control Pentasa (19.1%,
80.1% vs 100,0%, p=0.03). This promising result led us to
prepare new CB2 selective agonists,15 around the 4-oxo-
1,4-dihydropyridine template, with improved in vivo ac-
tivity.
Table 1. C-3 and C-6 modulations: affinities and func-
tional activities of 15-19 and 30-37 toward both canna-
binoid receptors.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Binding affinity
[
35S]-GTPγS (hCB2)
Ki (nM)
Cpd
R2
R4
EC50
(nM)
Emax
(%)
hCB2
hCB1
Chart 1. Modest effect of compound 10 against exper-
imental colitis.
tert-butyl
tert-butyl
tert-butyl
10.2 ± 2.2
9.0 ± 2.0
9.6 ± 0.7
> 1000
49.2 ± 6.6
47.1 ± 7.1
452 ± 77
191 ± 11
258 ± 6
254 ± 16
15
16
17
548
562
100
*
*
50
0
tert-butyl
tert-butyl
14.1 ± 1.8
9.2 ± 0.6
176
27.8 ± 9.2
8.3 ± 0.7
195 ± 11
250 ± 9
18
19
87.9
methyl
methyl
15.2 ± 3.7
6.4 ± 1.9
> 1000
> 1000
53.1 ± 7.7
254 ± 23
233 ± 15
30
31
84.2 ± 6.6
methyl
0.26 ± 0.17
> 1000
38.4 ± 7.0
216 ± 12
32
In vitro Pharmacology. The affinities of target com-
pounds 15-19, 30-45 and 47-54 (table 1-2) against the hu-
man cannabinoid receptors (hCB1 and hCB2) were deter-
mined by a competitive radioligand displacement assay
using [3H]-CP-55,940 as radioligand for both hCB2 and
hCB1 receptors.16 All compounds were first screened at 1
µM concentration and Ki values were determined for
those exhibiting a specific displacement superior to 60%
either for hCB1 or the hCB2 (tables 1-2). In addition, we
investigated their functional activity at CB2 receptor using
a [35S]-GTPγS binding assay and hCB2-CHO cells mem-
branes (tables 1-2).17
methyl
methyl
methyl
24.4 ± 2.3
> 1000
> 1000
49.9 ± 6.7
197 ± 3
33
34
35
/
/
/
/
/
/
/
/
/
> 1000
N
methyl
methyl
> 1000
36
37
> 1000
/
/
/
(R) – (+) WIN 55,212-2
3.1 ± 1.2
52.4 ± 1.2
89.6 ± 0.8
232 ± 2
Surprisingly, for compounds 18 and 19, when the tert-
butyl group at C-6 was replaced by a methyl (34 and 37),
the affinity for the CB2 receptor was completely lost. The-
se first results clearly suggest that, to achieve a good affin-
ity and selectivity at CB2, the methyl at C-6 as well as the
adamantyl group borne by the amide function should be
retained.
Starting from compound 10, first optimizations were
focused on the moiety borne by the amide at C-3 position,
keeping either tert-butyl (15-19, table 1) or methyl (30-45,
tables 1 and 2) group constant at the C-6 position. For
compounds presenting a tert-butyl group at C-6, and re-
gardless of the amide substituent, all evaluated com-
pounds (15-19) possess very good affinity (ca. 10 nM) for
the CB2 receptor. For instance, replacing the bulky ali-
phatic adamantyl group by methylcyclopropyl (18) or
phenyl (19) did not impact on CB2 receptor affinity. On
the other hand, these modifications led to compounds
with good affinity at CB1 (hCB1: Ki = 176 nM for 18 and Ki =
88 nM for 19) and therefore, to less CB2 receptor-selective
Next, we modified the n-pentyl chain at N-1 position by
other alkyl chains (table 2). Thus, we synthesized com-
pound 42 featured by a tetrahydropyranylmethyl group
that was shown to be important for the binding of indole
series.18 Even though this modification was tolerated, the
replacement of the tetrahydropyranylmethyl group by
other alkyl ether chains, resulted in a decrease (43), or
even loss (39-41, 44), of the affinity. Similarly, compound
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ished. Surprisingly, when the p-methyl was replaced by an
38 characterized by an unsaturated alkyl chain, displayed
1
2
3
4
5
6
7
8
no affinity at CB2 receptor. In addition, the introduction
of a trifluorobutyl chain (45) led to one of the most potent
and selective ligands within this series (hCB2: Ki = 0.28
nM, S.I > 3571).
ethyl (49), the CB2 activity was restored. Finally, a pyri-
dine heterocycle (48) at C-5 was well tolerated with a Ki
of 36 nM and no activity at CB1 at 1 µM.
With regard to their functional activity, all compounds
dose-dependently increased the [35S]-GTPγS binding be-
tween 188% to 258% (control value set at 100%), with EC50
values in between 8.3 and 452 nM, which indicates that
they behave as full agonists at CB2 receptor. Interestingly,
while we previously showed that introducing an aromatic
ring at C-6 position generally led to an inverse agonist
profile,13 the same kind of modifications at C-5 had no
impact on the functional activity, since all C-5 substituted
compounds show an agonist profile.
Table 2. N-1 and C-5 modulations: affinities and func-
tional activities of 38-45 and 47-54 toward both can-
nabinoid receptors.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Binding affinity
[
35S]-GTPγS (hCB2)
Ki (nM)
Cpd
R2
R4
In vivo Pharmacology. A set of target CB2 agonists,
characterized by high CB2 receptor affinity (hCB2 Ki < 50
nM) and a selectivity index of at least 10 (15-18, 30-33, 42,
45 and 48), was selected to be evaluated in the mouse
model of TNBS-induced colitis.14 Thus, each compound
was administered intraperitoneally once daily at the dos-
age of 10 mg/kg, starting three days before colitis induc-
tion. Pentasa granules mixed in food at the dosage of 150
mg/kg, and provided ad libitum during all the course of
the experiment, was used as anti-inflammatory positive
control (chart 2).
EC50
(nM)
Emax
(%)
hCB2
hCB1
38
39
H
H
> 1000
> 1000
/
/
/
/
/
O
/
/
O
O
40
41
H
H
> 1000
> 1000
/
/
/
O
/
/
O
O
42
43
44
H
H
24.1 ± 6.5
> 1000
47.4 ± 7.7
224 ± 6
O
Chart 2. The new CB2 receptor selective agonists at-
tenuate colitis in TNBS-induced colitis (data are the
mean ± SEM of 10 mice per group; *p < 0.05; **p< 0,01 vs
TNBS and vehicle).
195.8 ±
41.9
/
/
/
/
/
/
O
O
H
> 1000
45
47
48
H
I
0.28 ± 0.18
20.8 ± 6.3
36.5 ± 0.6
> 1000
193.4
17.3 ± 7.7
11.9 ± 0.8
114 ± 7
224 ± 11
299 ± 4
213 ± 36
CF3
C5H11
C5H11
100
N
*
*
> 1000
**
**
*
**
**
**
49
50
C5H11
C5H11
54.3 ± 5.4
> 1000
> 1000
39.9 ± 8.3
199 ± 12
**
**
**
50
0
**
/
/
/
O
51
C5H11
C5H11
13.4 ± 0.6
105.1
/
88.5 ± 7.9
/
188 ± 7
/
52
189.4 ± 71.7
53
54
C5H11
C5H11
> 1000
> 1000
3.1 ± 1.2
/
/
/
/
/
/
NC
(R) – (+) WIN 55,212-2
52.4 ± 1.2
89.6 ± 0.8
232 ± 2
Mice were euthanized three days after TNBS admin-
istration and parameters reflecting the degree of colon
inflammation were assessed. The colon of each mouse
was examined and damages were assessed by a semi-
quantitative scoring system. Mice that received the TNBS
showed macroscopic colitis reflected by thickening of the
bowel and ulceration areas. In this experiment, the posi-
tive control Pentasa was able to decrease colitis macro-
scopic scores by 28.2% (71.8% vs 100%).
To complete the SAR, we investigated the impact of the
C-5 substituent by introducing iodine, a cyclopropyl as
well as various aromatic groups at this position (47-54,
table 2). Generally, this modification led to less potent
and selective compounds. Introduction of iodine (47) and
cyclopropyl (51) allowed to maintain a high affinity at CB2
but decreased selectivity, while a phenyl group (52) signif-
icantly decreased CB2 affinity. When this phenyl group
was substituted in para position by a methyl group (53) or
in meta position by a cyano (54) or a methylketone group
(50), the affinity at CB2 receptor was completely abol-
Compounds 18, 31-33, did not provide better results
than the initial lead 10 (74.9-84.9% of colitis macroscopic
scores attenuation). Compounds 15, 42 and 45 decreased
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macroscopic scores with the same magnitude than Pen- Unfortunately, compound 48 failed to increase meta-
1
2
3
4
5
6
7
8
tasa (62.2%, 66.3%, 65.1%, respectively), while 16, 17 and
30, were shown to be more potent (60.0%, 51.5%, 55.9%,
respectively). Finally, compound 48 exerted a very strong
protecting effect against colitis as indicated by the 61.4%
decrease in colitis macroscopic score (38.6%). Because 48
was significantly more potent than the reference treat-
ment we sought to better characterize its properties.
Thus, histological damages, MPO activity as well as TNFα
and Il-1β mRNA were quantified. As can be seen from
chart 3, compound 48 significantly attenuated both histo-
logical scores (3.1 vs 4.9) and MPO activity (34.1% vs
73.7%) but no significant effect was observed on the
mRNA expression of the two cytokines TNFα and Il-1β.
bolic stability in human and mouse liver microsomes
since both 10 and 48 were rapidly metabolized by mouse
(Clint >200 µL.min-1.mg-1) and human (1% of parent reman-
ing after 60 min) hepatic microsomes.
Table 3. Determination and evaluation of selected
physicochemical and in vitro DMPK parameters.
9
Compound
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Parameters
MW (g/mol)
cLogPa
398.58
5.05
433.59
3.55
tPSA (Å2)a
49.4
61.8
In vitro DMPK
Chart 3. Histological damages (A), MPO activity (B),
TNFα and IL-1β mRNA levels (C, D) were measured in
colon after TNBS-induced colitis and treatment with
Pentasa (150 mg/kg) or compound 48 (10 mg/kg) (data
are the mean ± SEM of 10 mice per group; *p < 0.05 vs
TNBS and vehicle).
Human PPB (% free)b
1
7
A-B Caco-2 permeability (x 10-6 cm.s-1)c
P-gp inhibition (% at 10 µM)d
9.8
94.5
49.9
33.7
Metabolic stability (human liver microsomes,
% parent remaining after 60 min)e
1
>200
b
1
Mouse Clint (µl.min-1. mg-1)f
>200
aDetermined with ChemDraw Ultra 10.0. Assessed by equilibrium di-
alysis at 37 °C. Compounds were incubated (0 and 60 min) at 10 ꢀM in
Caco-2 cell line. Performed on MDR1-MDCKII cell line, using cellular
c
d
e
uptake of calcein AM. Compounds were incubated (0 and 60 min) at 1
ꢀM in human liver microsomes (0.3 mg/mL). fCompounds were incubated
at 1 ꢀM in mouse liver microsomes (0.3 mg/mL)
Even though generally, 48 exhibited an improved in
vitro DMPK profile relative to 10, its low metabolic stabil-
ity could represent a hindrance to oral administration.
CONCLUSION
The present study confirms that the 4-oxo-1,4-
dihydropyridine scaffold is suitable for an easy access to
potent CB2 receptor selective ligands. We have synthe-
sized and evaluated, on both cannabinoid receptors,
twenty nine new compounds characterized by different
moieties at N-1, C-3, C-5 and C-6 positions, allowing the
description of sharp SAR and the discovery of very potent
CB2 selective agonists. Among them eleven were evaluat-
ed in the TNBS-induced colitis model in mice and were
found active in this experimental model. Compound 48
emerged as credible lead in this series with a strong pro-
tecting effect against colitis. Though this compound failed
to act on TNFα and Il-1β levels, it has drastically de-
creased macroscopic scores, histological damages and
MPO activity. Future optimizations should be focused on
improving DMPK profile of this series of compounds in-
cluding metabolic stability.
Preliminary in vitro DMPK profile.
Initial lead 10 as well as 48 were profiled for key physi-
cochemical properties, in vitro metabolic stability, plasma
protein binding and intestinal absorption, allowing a
comparison between these two compounds (table 3).19
With a clogP of 5 and a tPSA below 50 Å2, initial lead 10
displayed a high lipophilicity while having an acceptable
molecular weight. Relative to 10, compound 48 resides in
less lipophilic space with a cLogP of 3.5 and a TPSA of 62,
while keeping a reasonable molecular weight.
EXPERIMENTAL SECTION
General Procedure for the Preparation of Com-
pounds 48-54. Compound 47 (1 equiv), appropriate bo-
ronic acid (1.5 equiv), potassium phosphate (4 equiv),
tricyclohexylphosphine (0.5 equiv) and palladium acetate
(0.25 equiv) were suspended in a mixture of toluene/H20
(3:2). The mixture was refluxed for 12 h and the palladium
acetate filtered. The resulting filtrate was then concen-
trated and the residue partitioned in H2O-CHCl3. The
organic phase was washed both with water and brine,
dried and evaporated. The crude material was chroma-
As for in vitro assessments (table 3), plasma protein
binding assay showed higher free levels for 48 than for 10
(7% vs 1%) and an improved permeability was measured
for 48 as compared to 10 in the in vitro CACO-2 assay.
Moreover, in contrast to compound 10, no interaction
with P-gp was attributed to 48 as evidenced from the cal-
cein-AM functional assay.
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Journal of Medicinal Chemistry
(6) D'Argenio, G.; Valenti, M.; Scaglione, G.; Cosenza, V.; Sor-
tographied on silica gel (DCM/MeOH 95:5, v/v) to afford
1
2
3
4
5
6
7
8
rentini, I.; Di Marzo, V. Up-Regulation of Anandamide Levels as
an Endogenous Mechanism and a Pharmacological Strategy to
Limit Colon Inflammation. FASEB J. 2006, 20, 568-570.
(7) Structures of compounds 1-9 shown in Supporting Infor-
mation.
target compounds 48-54.
N3-(1-Adamantyl)-6-methyl-4-oxo-1-pentyl-5-
(pyridin-4-yl)-1,4-dihydropyridine-3-carboxamide
(48). White solid (70%); mp > 250 °C; H NMR (CDCl3) δ
10.00 (s, 1H), 8.70 (d, 2H, J = 5.5 Hz), 8.51 (s, 1H), 7.16 (d,
2H, J = 5.8 Hz), 3.96 (t, 2H, J = 7.7 Hz), 2.19 (s, 3H), 2.11 (m,
9H), 1.69 (m, 8H), 1.38 (m, 4H), 0.94 (t, 3H, J = 6.5 Hz).
LC-MS (APCI+) m/z 434.3 (MH+). HRMS calcd for C27 H36
O2 N3 434.2802, found 434.2788.
1
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Monory, K.; Cravatt, B. F.; Ferri, G. L.; Sibaev, A.; Storr, M.; Lutz,
B. The Endogenous Cannabinoid System Protects Against Colon-
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J. M.; Molleman, A.; Pertwee, R. G.; Parsons, M. E. The Effects of
Delta-Tetrahydrocannabinol and Cannabidiol Alone and in
Combination on Damage, Inflammation and In Vitro Motility
Disturbances in Rat Colitis. Br. J. Pharmacol. 2010, 160, 712-723.
(9) Kimball, E. S.; Schneider, C. R.; Wallace, N. H.; Hornby, P.
J. Agonists of Cannabinoid Receptor 1 and 2 Inhibit Experimental
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um. Am. J. Physiol. Gastrointest. Liver Physiol. 2006, 291, 364-371.
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katti, M.; Nagarkatti, P. S.; Cannabinoid Receptor-2 (CB2) Ago-
nist Ameliorates Colitis in IL-10(-/-) Mice by Attenuating the
Activation of T Cells and Promoting their Apoptosis. Toxicol.
Appl. Pharmacol. 2012, 258, 256-267.
(11) (a) Storr, M. A.; Keenan, C. M.; Emmerdinger, D.; Zhang,
H.; Yüce, B.; Sibaev, A.; Massa, F.; Buckley, N. E., Lutz, B., Göke,
B.; Brand, S.; Patel, K. D.; Sharkey, K. A. Targeting Endocanna-
binoid Degradation Protects Against Experimental Colitis in
Mice: Involvement of CB1 and CB2 Receptors. J. Mol. Med. (Berl)
2008, 86, 925-936. (b) Andrzejak, V.; Muccioli, G. G.; Body-
Malapel, M.; El Bakali, J.; Djouina, M.; Renault, N.; Chavatte, P.;
Desreumaux, P.; Lambert, D. M.; Millet, R. New FAAH Inhibitors
Based on 3-Carboxamido-5-Aryl-Isoxazole Scaffold that Protect
Against Experimental Colitis. Bioorg. Med. Chem. 2011, 19, 3777-
3786.
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ASSOCIATED CONTENT
Structures of endocannabinoid system-interacting ligands
with protective effect against colitis, experimental proce-
dures and spectroscopic data for all synthesized compounds,
and detailed pharmacology. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Pr. Régis Millet: Phone: +33320964374. Fax: +33320964906.
E-mail: regis.millet@univ-lille2.fr.
Present Address
† Bioanalysis and Pharmacology of Bioactive Lipids Lab,
Louvain Drug Research Institute, Université Catholique de
Louvain, 72 Avenue E. Mounier. B1.72.01, B-1200 Bruxelles,
Belgium.
ACKNOWLEDGMENT
We are grateful for the financial support from ANR emer-
gence (ANR-09-EBIO-007: CAB-MICI) and PRIM (Pole de
Recherche Interdisciplinaire pour le Medicament).
(12) Alhouayek, M.; Lambert, D. M.; Delzenne, N. M.; Cani, P.
D.;
Muccioli,
G.
G.
Increasing
Endogenous
2-
Arachidonoylglycerol Levels Counteracts Colitis and Related
Systemic Inflammation. FASEB J. 2011, 25, 2711-2721.
(13) El Bakali, J.; Muccioli, G. G.; Renault, N.; Pradal, D.; Body-
Malapel, M.; Djouina, M.; Hamtiaux, L.; Andrzejak, V.;
Desreumaux, P.; Chavatte, P.; Lambert, D. M.; Millet, R. 4-Oxo-
1,4-Dihydropyridines as Selective CB2 Cannabinoid Receptor
Ligands: Structural Insights into the Design of a Novel Inverse
Agonist Series. J. Med. Chem. 2010, 53, 7918-7931.
(14) For experimental procedure, see Supporting Information.
(15) For synthetic details, procedures and compound charac-
terization, see Supporting Information.
(16) (a) Govaerts, S. J.; Hermans, E.; Lambert, D. M. Compari-
son of Cannabinoid Ligands Affinities and Efficacies in Murine
Tissues and in Transfected Cells Expressing Human Recombi-
nant Cannabinoid Receptors. Eur. J. Pharm. Sci. 2004, 23, 233-
243. (b) For experimental procedure, see Supporting Infor-
mation.
(17) (a) Govaerts, S. J.; Muccioli, G. G.; Hermans, E.; Lambert,
D. M. Characterization of the Pharmacology of Imidazolidinedi-
one Derivatives at Cannabinoid CB1 and CB2 Receptors. Eur. J.
Pharmacol. 2004, 495, 43-53. (b) For experimental procedure, see
Supporting Information.
(18) Frost, J. M.; Dart, M. J.; Tietje, K. R.; Garrison, T. R.; Gray-
son, G. K.; Daza, A. V.; El-Kouhen, O. F.; Yao, B. B.; Hsieh, G. C.;
Pai, M.; Zhu, C. Z.; Chandran, P.; Meyer, M. D. Indol-3-
ylcycloalkyl Ketones: Effects of N1 Substituted Indole Side Chain
Variations on CB(2) Cannabinoid Receptor Activity. J. Med.
Chem. 2010, 53, 295-315.
ABBREVIATIONS
IBD, inflammatory bowel disease ; CNRs, cannabinoid re-
ceptors; ECs, endocannabinoids; AEA, anandamide; 2-AG, 2-
arachidonoylglycerol; GI, gastrointestinal; TNBS, 2,4,6-
trinitrobenzenesulfonic acid; MPO, myeloperoxidase; Il-1β,
interleukin-1β; SAR, structure-activity relationship; CHO,
Chinese hamster ovary; hCB1&2, human cannabinoid receptor
1 & 2
REFERENCES
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(19) Studies performed at Cerep according to their internal
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4-Oxo-1,4-dihydropyridines as Selective CB2 Cannabinoid Receptor Ligands Part 2: Dis-
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Jamal El Bakali, Pauline Gilleron, Mathilde Body-Malapel, Roxane Mansouri, Giulio G. Muccioli, Madjid
Djouina, Amélie Barczyk, Frédérique Klupsch, Virginie Andrzejak, Emmanuelle Lipka, Christophe Fur-
man, Didier M. Lambert, Philippe Chavatte, Pierre Desreumaux, and Régis Millet.
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